The proposed project involves an in-depth study of the mechanisms responsible for NaCl absorption and for cell volume regulation by the in vitro microperfused mouse medullary thick ascending limb of Henle's loop (mTALH). These studies will employ two distinct methodologies. First, electrophysiological techniques will be employed to: (a) characterize the K+ conductive pathway in apical plasma membranes with particular emphasis on blockade by protons; these data may provide important information on the nature of the organic groups in the transport pathway critical to K+ movement, and should permit evaluation of the effects of ADH on the proton-blocking characteristics of the K+ conductance. (b) Determine the instantaneous current-voltage (I-V) relations of apical and basolateral cell membranes for K+ and Cl- both with and without ADH; these data should permit explicit evaluation of the effects of ADH on the K+ and Cl- permeabililties and chrod conductances of individual cell membranes and on the intracellular activities of K+ and Cl-. (c) Evaluate the hypothesis that the ADH-mediated increase in cell conductance, that accompanies the hormone-induced increases in NaCl absorption, is due to the recruitment of K+ conductance units from a pool not originally present in apical membranes. Second, a unique computer-assited optical technique, coupled with electrophysiological techniques, will be used to: (d) characterize the transport mechanisms that mediate cell volume regulation in response to hypertonic peritubular media, (e) evaluate the mechanism responsible for activation of hypertonic volume regulation by the mTALH anad (f) evaluate the effects of increased in peritubular osmolality, and the attendant compensatory volume regulation, on the transport processes responsible for active NaCl absorption. The results of these studies will provide a more basic understanding of urinary concentrating mechanisms and the role of peritubular osmolality, and the associated cell volume regulation, in modulating these processes; and provide insights into how the mTALH, as one of several different nephron segments in the medulla, controls cell volume during increases in interstitial osmolality attendant to antidiuresis.